High-throughput Raman-activated cell sorting of microalgal genome-wide edited library revealed a new regulatory pathway for carotenoid synthesis

Abstract

Abstract Functional genomics has been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated Cell Sorting (RACS). The CRISPR/Cas-generated library consists of 3,567 microalgal mutants derived from 2,397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unravels mutations in the violaxanthin de-epoxidase ( noVDE ) or in the proteasome assembly chaperone 4 ( noPAC4 ) genes. Knocking out all five known noVDE s reveal that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidences suggest two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this new forward-genetics approach can greatly accelerate the discovery of new genes and pathways.

Code and data availability

The paper's authors' analysis code (scmli, including a modified snippy) is publicly available on GitHub, qualifying as a paper-specific computational asset. The validated gRNA/mutant library data at NanDeSyn is stated to be 'freely available upon request', so it is request_only rather than public. Generic pipelines (nf